Jacobs:Protocol NuPAGE Electrophoresis and Western Blotting for Proteins

Materials

• Protein sample
• Protein Ladder
• Deionized water (autoclave 3L of milipore water)
• NuPAGE® LDS Sample Buffer
• NuPAGE® Reducing Agent
• NuPAGE® Novex Bis-Tris Gel: Gel Thcknss: 1.0 mm, Gel Sze: 8 cm x 8 cm, Load Volume: 25 μl
• NuPAGE® SDS Running Buffer
• Electrophoresis module
• Blotting membranes
• Filter paper
• Methanol (if using PVDF)
• XCell II. Blot module
• NuPAGE® Transfer Buffer
• NuPAGE® Antioxidant

Procedure

Running the Gel

1. Wear protective gloves when handling gels and solutions
2. Determine amount of sample needed for x µg of protein in each electrophoresis sample
3. Turn on heating block to 70°C about 15 minutes before step 6.
4. Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer(20X) as follows in 1000mL Pyrex Bottle and mix thoroughly, Running Buffer:

1. NuPAGE® SDS Running Buffer (20X MOPS) 50 ml 2. Deionized Water 950 ml 3. Total Volume 1000 ml • Set aside 800 ml of the 1X NuPAGE® SDS Running Buffer for use in the lower (Outer) Buffer Chamber of the XCell SureLock Mini-Cell • 200 ml of 1X NuPAGE® SDS Running for use in the Upper (Inner) Buffer Chamber of the XCell SureLock. Mini-Cell. • Keep at room temperature

1. Prepare reducing sample in this order (Total volume depends on strength of protein):

1. Sample x μl 2. NuPAGE LDS Sample Buffer (4X) 4°C 3. NuPAGE® Reducing Agent (10X) 4°C 4. Maximum Total Volume 25 μl 5. Notes: For reduced sample, add the reducing agent immediately prior to electrophoresis to obtain the best results. The presence of more glycerol also increases the viscosity of the NuPAGE® LDS Sample Buffer. By bringing the NuPAGE® LDS Sample Buffer to room temperature (25°C), the buffer is more manageable.

1. Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 70°C for 10 minutes for optimal results.
2. Remove the NuPAGE® Gel from the pouch
3. Rinse the gel cassette with deionized water 2x. Peel off the tape from the bottom of the cassette.
4. In one smooth motion, gently pull the comb out of the cassette
5. Fill the sample wells with 1X NuPAGE® SDS Running Buffer using loading tips to rinse. Invert the gel and shake to remove the buffer 2x. Load buffer into wells one more time to push all bubbles out.
6. Orient the two gels in the Mini-Cell such that the notched well-side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.
7. If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette
8. Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.
9. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer (200mL). The buffer level must exceed the level of the wells.
10. Load an appropriate volume of sample at the desired protein concentration onto the gel
11. Load appropriate protein molecular weight markers
12. Fill the Lower (outer) Buffer Chamber with 600 ml of 1X NuPAGE® SDS Running Buffer
13. Immediately run the gel at 60 mA/gel, max 100 V. Run for 50-55 minutes until samples run onto the foot of the gel. (Check for bubbles emerging from solution to see if electrophoresis is running.)
14. Refer to step 1 and 2 of Western Blotting

Removing the Gel after Electrophorsis

1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock. Mini-Cell
2. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (well) side of the cassette should face up.
3. Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated
4. Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate

Western Blotting

1. Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows in 1000mL Pyrex bottle: Transfer Buffer:

1. NuPAGE® Transfer Buffer (20X) 50 ml at room temp 2. Methanol (100 mL for 1 gel) (200mL for 2 gels) 3. Deionized Water (849 mL for 1 gel) (749 mL for 2 gels) 4. Total Volume 1000 ml 5. Put in 4°C

1. Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturated during electrophoresis. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed.
2. Put 650mL dH20 into 4°C for step 21.
3. Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwich. Keep track of shiny side of transfer membrane
4. Soak the filter paper and transfer membrane briefly in 1X NuPAGE® Transfer Buffer immediately prior to use.
5. Use the gel immediately following the run. Do not soak the gel in running buffer
6. After prying opening the gel cassette remove wells with the Gel Knife. May run some transfer buffer over surface to remove air bubbles.
7. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller
8. Turn the plate over so the gel and filter paper are facing downwards over parafilm or clean flat surface
9. Use the Gel Knife to push the foot out of the slot in the plate and the gel will peel off
10. When the gel is on a flat surface, cut the foot off the gel with the gel knife
11. Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane shiny side down on the gel, ensuring all air bubbles have been removed
12. Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles
13. Place two soaked blotting pads into the cathode (-) core of the blot module. The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on blotting pad in the same sequence, such that the gel is closest to the cathode core (follow manual)
14. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components
15. Position the gel/membrane assembly and blotting pads in the cathode core of the XCell II. Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place
16. Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber
17. Place the Gel Tension Wedge so that its vertical face is against the blot module. Lock the Gel Tension Wedge by pulling the lever forward
18. Fill the blot module with 1X NuPAGE® Transfer Buffer until the gel/membrane assembly is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat
19. Fill the Outer Buffer Chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run
20. Place the lid on top of the unit
21. With the power turned off, plug the red and black leads into the power supply. Run the transfer at 10 Volts with an expected current of 100mA for 24 hours in 4°C in an ice bucket or for about 75 minutes room temp

Immunoblot Assay

1. Rinse membrane twice with dwater
2. Block membrane 20min-2hours (depending on protein) at room temperature with agitation in freshly prepared 5% albumin (Catalog 20-200) in TBS (TBS-MLK)
3. Incubate membrane overnight at 4°C with agitation and anti-x, diluted 1:2000 in freshly prepared TBS-MLK (dilution depends on protein)
4. Wash membrane twice (5-10min each) with TBS-Tween
5. Incubate membrane for 1hr at room temperation with agitation in anti-goat-HRP conjugate (1:2000 dilution in TBS) (dilution depends on protein) and Bio-rad precision protein Strep Tactin-HRP (1:30,000 dilution in TBS-Tween binds to protein ladder)
6. Wash membrane 3x (5-10min each) with TBS-Tween

Chemiluminescence Detection

1. Use Bio-rad Immu-star WesternC Chemiluminescent Kit. Mix 6 mL of Detection Reagent 1 and 2 for each transfer membrane (1:1).
2. Pour over transfer membrane and leave on for one minute.
3. Dry off with Kimwipe
4. Bring to the imager. Settings:
5. Exposure Type: Precision
6. Exposure Time: Auto
7. Sensitivity: Standard/High
8. Method: Chemiluminescence
9. Adjust brightness and focus >> Capture Image and save

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• Last updated 3/1/07